The present invention relates to an apparatus and a process for determining the rheological properties of biological fluids, and particularly for determining the filterability of cells in suspension and the viscosity of biological liquids. It relates more particularly to an apparatus and a process for determining the deformability of the red corpuscles of the blood. The biconcave and disk-like lens shape of the red corpuscle ensures for this latter (which has a diameter of about 7.5.mu.), the elasticity or the deformability required to allow it to pass through the circulatory ducts of the organism some of which scarcely exceed 2 to 3.mu. in diameter). The role of the deformability of the red corpuscles in circulatory and more precisely microcirculatory physiology, is now well-known (sometimes, it is the only means for detecting certain anomalies of the blood which are at the origin of different illnesses) and so it is very important to be able to measure this deformability. Further, this measurement (necessarily made in vitro) must accurately reflect and exactly translate this important quality of the red corpuscle, it must be accurate, reliable and reproducible, and it must be also as simple and as economical as possible, so as to be able to be used on a large scale not only in laboratories for biological analyses but also in research laboratories.
Numerous methods have been proposed for determining and measuring the deformability of the red corpuscle. Thus, the following have been used:--measurements of suction and flow in capillary tubes and micropipettes;--viscosimetric techniques;--microscopic examinations, after transformation of the erythrocytes by means of chemical agents;--measurements by light diffraction;--compression studies of the centrifugation button;--sedimentation and centrifugation backing measurements;--studies of the extension of the fixed corpuscles, on a microthread;--and the filtration or more exactly the filterability of the red blood corpuscles.
For example, the following have been advocated:
measurement of the deformability by the suction technique through a micropipette [RAND and BURTON (Bioph. J. 4, 115 1964)], by means of which the length of the "finger" of the membrane of the sucked-up corpuscle is measured. Though insofar as the principle and results are concerned, this technique gives satisfaction, this measurement is still only practised exceptionally because of its difficulty and its duration which is rather long; PA1 viscosimetric measurements (by means of a capillary or rotary viscosimeter). PA1 techniques of microscopic examination of the corpuscles; these techniques concern the microscopic examination of the cells in a flow of the Poiseuille type (for example in a capillary or in a transparent cone-flat viscosimeter). It is a very delicate technique which requires complex apparatus and whose results may sometimes be unreliable: the cells must in fact be placed in suspension in a very viscous nonphysiological medium (compare the "Rheoscope" by SCHMID-SCHONBEIN). PA1 techniques of indirect visualization of the deformation by studying the images of the diffraction of light. PA1 methods based on filtration. PA1 A: surface tension of the liquid PA1 .theta.: angle of connection between the liquid and the internal wall of the capillary. If .theta.=0, the liquid damps perfectly the solid; if .theta.=180.degree., the damping is zero. Angle .theta. depends on the nature of the liquid and of the capillary; for plastic materials (such as polycarbonate) and water, .theta. is greater than 90.degree.. PA1 Generally, the membrane is placed on the support. This stops up a fraction .alpha. of the pores, so that the number of pores really effective at time t.sub.o is (1-.alpha.)N.sub.o. It is possible, in some cases, that .alpha. differs from one experiment to another (for example because of puckering of the membrane, or of deformations of the support, or of the equality of this latter). PA1 The result will then be variations of the filtration times, all else being equal. PA1 Some cells of the suspension may clog up the pores of the filter. This results in progressively reducing (1-.alpha.)N.sub.o. PA1 In other words, the flow rate is a decreasing function of time. If c' is the concentration of the cells in question, and in assuming in Poiseuille's relationship (4), all other factors except N.sub.o constant, we will then write D=K.sub.1 N. PA1 If we admit that a pore is clogged up each time that a cell is engaged therein, the number of pores which clog up during time dt will be dN=c'dV or EQU dN=c'dt PA1 a three-way cock communicating the central duct of the lower block either with an overpressure ballast-receptacle, or with a "measure" circuit; PA1 a ballast-receptacle of a capacity between 0.1 and 5 liters, provided with a device for creating a small overpressure (0 to 50 cm of water) which may be a syringe or a pear-shaped bulb fitted with a valve, a receptacle filled with liquid which is raised, or similar; PA1 a "measure" circuit formed: PA1 either by a simple tube open to the atmosphere, if it is desired to filter only with the hydrostatic pressure of the liquid filling the capillary of the upper block, PA1 or by a tube connected to a second ballast-receptacle of 0.1 to 5 liters, having a vacuum inlet pipe if it is desired to filter with a depression. Here again the partial vacuum may be obtained by means of a syringe or a pear-shaped bulb fitted with a valve, a receptacle filled with liquid which is lowered, or similar.
Generally, all the measurements advocated [compare in particular the work of CHIEN et alia, Biorheology 12, 341, 1975; of THURSTON (Biophys. J. 12, 1205, 1972) and of USAMI et alia (Biorheology 10, 425, 1973)] are based on the concept according to which the viscosity of suspensions measured at a high speed gradient is smaller when the particles are deformable. However, such determinations are not very sensitive and very often require costly apparatus not very convenient to handle.
The results are also difficult to interpret, even after having fixed the shape of the cells by means of chemical agents such as glutaraldehyde or acetaldehyde.
This technique, studied more particularly by BESSIS and MOHANDAS [Blood Cells. 3 229-239 (1977) and Blood Cells 1 307-313 (1975)] also measures the extension of the cells subjected to shearing stresses, by using the diffraction of light (and particularly the diffraction of a laser ray passing through the blood sample).
These methods of measurement, besides being slow and not very well adapted to industrial measurements, require furthermore very costly apparatus whose handling is delicate: the cells must always be placed in a very viscous medium, very different from the physiological conditions.
Among the very numerous methods used for estimating the erythrocytary deformability, filtration is perhaps the only one which, up to date, has known a great extension, probably because of its simplicity.
The general principle for determining the erythrocytary filterability is the measurement of the flow of a suspension of red corpuscles concentrated to a greater or lesser degree through a membrane whose pores have a mean diameter less than that of the red blood corpuscles. For a given membrane and motive pressure, the filtration flow will be all the smaller the more difficult it is for the red blood corpuscles to be deformed.
The filtration flow is given by: EQU D=dV/dt (1)
where dV is the volume of liquid passing through the filter during the time dt. If this latter is not small enough for the flow to be constant, the measured flow is a mean flow given by: EQU D=.DELTA.V/.DELTA.T (2)
Generally, the mean flow measured between two times t.sub.2 and t.sub.1 (such as t.sub.2 -t.sub.1 =.DELTA.t) will be expressed by the relationship (3) below: EQU D=1/.DELTA.t Ddt (3)
An examination of the relationship (3) shows that the mean and instantaneous flows are only identical when D is independent of time (constant flow rate). For a membrane, the filtration flow is the sum of the elementary flows through each pore of the membrane.
If d represents the value of the flow rate of an homogeneous fluid of viscosity .eta..sub.o through a pore of a porous membrane of unit area, and l the length of the axis of the pore (assumed perpendicular to the surface of the filtering membrane) which forms the thickness of this membrane, and if r is the radius of the pore, we have (Poiseuille's relationship) ##EQU1## P representing the pressure difference between the inlet and the outlet of the pore.
If N.sub.o represents the number of pores of the membrane at time t.sub.o, we then have: ##EQU2##
For example, for a typical membrane of the trademark "NUCLEPORE" (manufactured by the GENERAL ELECTRIC COMPANY)
(where
r=2.5.mu.
1=12.mu.
N.sub.o =4.10.sup.5 /cm.sup.2
and for a liquid such as water at 25.degree. C. (.eta..sub.o =0.01 poise), we have D.perspectiveto.1 cm.sup.3 /second for L cm.sup.2 of membrane.
Based on this data, a large number of methods and apparatus have been described for measuring the filtration or more exactly the filterability of red corpuscles. In particular:
S. CHIEN et alia [Biorheology 8, 163 (1971)] measure the percentage of cells which pass while applying a pressure gradient;
L. S. LESSIN et alia [Blood Cells 3, 241-262 (1977)] also use a positive pressure through the "NUCLEPORE" membrane while using a very complicated apparatus;
SCHMID-SCHONBEIN et alia [Blut (1973) 26, 369-379], and REID H. L. et alia [J. Clin. Pathol. (1976) 29(9), 855-858] use on the other hand a simple device allowing a vacuum (about 20 cm of water) to be ensured, to which is connected a filter-support able to receive a filtering "NUCLEPORE" membrane made from polycarbonate having a diameter of 13 mm, whose cylindrical pores have a diameter of 5.mu.. A 1 ml syringe may be adapted to the filter-support. The filterability of the red blood corpuscles in a 40% suspension in physiological serum is assessed by timing the time taken for 1 ml of suspension to pass;
P. TEITEL [Nature (Lond.) 184, 1808 (1959), Sangre (Barcelona) 9, 282 (1964), Blood Cells 3, 55-70 (1977)] advocates quite simply a paper filter (the pore diameters range between 20 and 40.mu.), the only pressure exerted being gravimetric, the measurement being effected by determining the flow rate of the liquid (a suspension of washed red blood corpuscles, whose hematocrit is greater than 90%) through the filter.
It should be noted that the use of the "NUCLEPORE" filter as a model for the capillary was already advocated in 1966 [GREGERSEN et alia "Hemorheology" (Copley Ed.) Pergamon Press. Oxford].
As can be seen from the above, all the techniques proposed in the prior art do not comply with the criteria which require an analysis to be at one and the same time simple to carry out, economical, accurate, reproducible and reliable, and to "measure" perfectly the properties of the red corpuscles represented by their flexibility, their elasticity and their deformability.
It is clear, in examining the relationship (4) above, that to have accurate, reliable and reproducible measurements and especially to have measurements which correspond to the reality of the facts, i.e. which are indeed connected with this essential mechanical quality of the red blood corpuscles formed by the deformability, a certain number of parameters must be reconsidered and restated.